The rapid pace of genetic research has required the development of new research tools to efficiently determine both genotype and gene expression levels in cellular organisms. “Gene chips” which contain arrays of short DNA or RNA chains in an array of sequences bound to a substrate (usually glass) are now commercially available. The chip is indexed so that the particular sequence bound in any area is known (or, in the case of cDNA, at least the cell line is known); a region having a homogeneous composition is referred to as a “feature.” The chips can be incubated with a target solution containing DNA or RNA bound to a fluorescent tag, allowing the binding of target DNA or RNA to individual features. Such systems can be used for the determination of both genotype and gene expression levels.
In genotype analysis, it is usually merely the presence or absence of binding that must be sensed. If fluorescence is observed above a threshold level in a particular region, binding has occurred and a sequence is identified by consulting the index of DNA or RNA positions on the chip. It is currently necessary for feature sizes to be large enough that their locations can be accurately identified by dead reckoning (possibly based on cued fluorescent features deposited at the same time as the feature array).
A more difficult problem is the quantitative measurement of levels of gene expression using DNA or RNA chip methods. The chemical density of a particular species is generally monotonically related to its level of fluorescence Thus, the intensity of the fluorescence can be measured to obtain information about the chemical density. A portion of an exemplary chip is shown in FIG. 1. Fluorescence levels may span 2-3 orders of magnitude in some cases; thus, determining the position of both bright and dim signals cannot generally be accomplished by simple calculations, such as thresholding of signal images. A variety of image analysis techniques exist for identifying feature locations for intensity measurement, but most rely on the feature array being perfectly regular, at most being able to make simple linear compensations for small amounts of stretching and rotation.
It is desirable to provide chips having small feature sizes, in order to increase the number of features that can be placed on a single chip. However, as feature sizes decrease, systematic errors in feature deposition and scanning may make accurate feature location by dead reckoning increasingly impractical. It is an object of the present invention to provide a superior system for correlating bright and dim regions of a scanned substrate with known underlying features in order to accurately measure feature intensity and position, thereby obtaining accurate analysis of the underlying signal for each feature.